Symbionts as Major Modulators of Insect Health: Lactic Acid and Honeybees

Alejandra Va´squez1*., Eva Forsgren2, Ingemar Fries2, Robert J. Paxton3,4, Emilie Flaberg5, Laszlo Szekely5, Tobias C. Olofsson1*. 1 Department of Laboratory Medicine, Medical Microbiology, Lund University, Lund, Sweden, 2 Department of Ecology, Swedish University of Agricultural Sciences, Uppsala, Sweden, 3 School of Biological Sciences, Queen’s University Belfast, Belfast, United Kingdom, 4 Institute for Biology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany, 5 Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden

Abstract Lactic acid bacteria (LAB) are well recognized beneficial host-associated members of the microbiota of humans and animals. Yet LAB-associations of invertebrates have been poorly characterized and their functions remain obscure. Here we show that honeybees possess an abundant, diverse and ancient LAB microbiota in their honey crop with beneficial effects for bee health, defending them against microbial threats. Our studies of LAB in all extant honeybee species plus related apid bees reveal one of the largest collections of novel species from the genera Lactobacillus and Bifidobacterium ever discovered within a single insect and suggest a long (.80 mya) history of association. Bee associated microbiotas highlight Lactobacillus kunkeei as the dominant LAB member. Those showing potent antimicrobial properties are acquired by callow honey bee workers from nestmates and maintained within the crop in biofilms, though beekeeping management practices can negatively impact this microbiota. Prophylactic practices that enhance LAB, or supplementary feeding of LAB, may serve in integrated approaches to sustainable pollinator service provision. We anticipate this microbiota will become central to studies on honeybee health, including colony collapse disorder, and act as an exemplar case of insect-microbe symbiosis.

Citation: Va´squez A, Forsgren E, Fries I, Paxton RJ, Flaberg E, et al. (2012) Symbionts as Major Modulators of Insect Health: Lactic Acid Bacteria and Honeybees. PLoS ONE 7(3): e33188. doi:10.1371/journal.pone.0033188 Editor: Niyaz Ahmed, University of Hyderabad, India Received January 20, 2012; Accepted February 9, 2012; Published March 12, 2012 Copyright: ß 2012 Va´squez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by grants from the Gyllenstierna Krapperup’s Foundation, Ekhaga Foundation, the Swedish Board of Agriculture, The Swedish Research Council Formas, the EU FP7 project BeeDoc (244956), the Biotechnology and Biological Sciences Research Council’s Insect Pollinators Initiative (grant BB/ I000100/1), and the European Science Foundation COST (European Cooperation in Science and Technology) network COLOSS (FA0803).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (AV); [email protected] (TO) . These authors contributed equally to this work.

Introduction bacterial group for the food industry and the fermentation of dairy products. In addition, strains within LAB are also generally Symbiosis is common in nature, in which symbionts as recognized as safe (GRAS) food grade microorganisms and commensals or mutualists evolved to benefit each other. Culture- employed as probiotics bestowing human health [10,11]. Genera independent studies of the human microbiota identified recently a within LAB are functionally related by phenotypic characteristics complex symbiotic environment with more than 1,000 bacterial [12] and considered as beneficial organisms commonly found as phylotypes representing more than 7,000 strains [1]. The both exogenous and endogenous microbes in healthy individuals. composition of this microbiota has been suggested to be a result LAB found within humans and animals as commensals are known of a highly coevolved symbiosis and commensalism influenced by to protect their hosts via antimicrobial metabolites and modulation nutrition, physiology and immunological factors [2,3]. of host immune response [13,14]. One of the most important The insect gut has been described as the greatest unexplored genus within LAB is Lactobacillus, which is continuously under reservoir of microbiological diversity [4]. Ryu and colleagues [5] taxonomic discussion and includes at present 175 listed species established the importance of the normal flora in the fruit fly gut in [15]. order to sustain health. This small microbiota was sufficient to We have discovered a rather special symbiotic lactic acid suppress growth of pathogens. While insects harbour a smaller bacterial (LAB) microbiota within the honey crop of the Western number of symbionts compared to humans they may be even honeybee Apis mellifera [16]. The crop is a central organ in the more important [6]. Studies have shown that symbiosis between honeybee’s food production between the oesophagus and social insects and microbial species are often highly coevolved [7] ventriculus, used for collection and transport of nectar to hive. and that these symbionts are evolutionary shaped distinctly from The crop microbiota of A. mellifera is composed of 13 bacterial the forces acting upon symbionts of solitary organisms, which species within the genera Lactobacillus and Bifidobacterium [16,17,18] normally lack a homeostatic fortress environment [8]. and it plays a key role in the production of honey [16] and bee- Lactic acid bacteria (LAB) are found as commensals within bread [19], long term stored food for both adult honeybees and humans, insects and animals [9]. They confer an important larvae. Our recent studies on the subspecies of A. mellifera have also

PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e33188 Symbiosis between LAB and Honeybees demonstrated that the LAB microbiota is consistent across its and fluorescence microscopy that the microbiota form biofilms native and introduced range [17]. and networks (Figure 2 and Figure 3; Video S1 and Video S2) by Metagenomics has been used to identify a rich diversity of which they attach to the wall of the crop using structures microbes within honeybees afflicted by Colony Collapse Disorder resembling extracellular polymeric substances (EPS) (Figure 2). (CCD) [20], including emergent pathogens (i.e. Nosema ceranae and viruses) [21], while recent studies have picked up novel bacterial Functional characterization genera within the intestinal tract of bees by culture independent The microbial composition of 15 flowers frequently visited by A. methods [20,22,23]. Some of these may comprise important mellifera in Sweden was investigated (Table S1). In total sixty symbionts for the maintenance of bee health; however, these transient microorganisms were isolated from flowers and identified descriptive methods do not inform on the functional role or to the species level (Accession nr: JN167926-JN167985). We tested importance of the bee crop microbiota or of individual symbionts the inhibition properties of all honey crop LAB grown individually within this niche. and together against the 55 bacterial strains and 5 yeast strains We have demonstrated by both in vitro and in vivo studies that the isolated from flowers. There was a clear overall inhibition of all LAB microbiota in A. mellifera inhibit one important honeybee transient flower microorganisms by single members of the LAB pathogen, the bacterial brood pathogen larvae that is microbiota in the honey crop (Table S1). L. kunkeei, the most the cause of the brood disease American foulbrood (AFB) [24]. In common species in all bees (Figure 1), was also the most potent, the current study we investigate if the LAB microbiota is consistent inhibiting all tested microorganisms. in all nine recognized honeybee (Apini) species plus stingless bee The LAB microbiota partly inhibited the bee pathogen M. species (Meliponini), a phylogenetically close taxon that, like plutonius in vivo (Figure 4) and totally in vitro, L. kunkeei and the honeybees, are eusocial, live in colonies comprising one queen and thirteen LAB together showed the best inhibition properties. The 100’s to 10,000’s of workers, store large quantities of honey and overall effect of adding the LAB mixture to bee larval food was a bee bread and are managed commercially or exploited by ethnic significant reduction in the number of dead larvae (Figure 4, 2 groups across the tropics. X 4 = 24.27, 2-tail P: ,0.001). The total mortality of the Functional characterization of the endogenous crop microbiota uninfected control groups (with or without LAB supplement) was is essential in providing insights for the understanding of its role for ,7% in both replicates. The results demonstrate that addition of bee health and disease. Here we explore the diversity, mainte- LAB to the food of young honeybee larvae exposed to M. plutonius nance and dynamics of LAB in the honey crop and the pivotal role decreases the number of larvae succumbing to EFB infection. that they play in bee health, with major implications for research on bee decline and sustainable pollinator management. LAB and antibiotics In vitro culturing of the 13 individual LAB members with two Results antibiotics (oxytetracycline and tylosin) used in apiculture to control bacterial diseases AFB and EFB demonstrated high LAB diversity sensitivity of all LAB members to Tylosin, the most recently All 9 Apis and the 3 Meliponini species studied possess a similar employed antibiotic within apicultural practices in the USA [26]. microbiota, comprising together approximately 50 novel LAB Nevertheless, strains L. kunkeei Fhon2 and Lactobacillus Fhon13, species in the genera Lactobacillus and Bifidobacterium (Figure 1), Hma11, Hma8 and Hon2 showed resistance to oxytetracycline. using the threshold used to define a bacterial species based on , , rRNA gene sequencing of 97% similarity [25] ( 98.5% is often Accession numbers found to imply possibly novel species). The 16S rRNA gene sequences generated in this study are One particular LAB was common and dominated the available from GenBank under the accession numbers: microbiota of Apis spp.: L. kunkeei (44% of 750 isolates were L. HM534742–HM534842, JN167926–JN167985 and JN689233. kunkeei). However, many Lactobacillus and Bifidobacterium were found to be common across bee species (Figure 1), including L. kunkeei in stingless bees from Central America (M. beecheii) and Africa (M. Discussion bocandei), though not in Trigona sp. from Borneo and Thailand, Our results reveal one of the largest collections of novel species where it is sympatric with five native Apis species. From our from the genera Lactobacillus and Bifidobacterium ever discovered previous studies [16,19] we know that bees add the LAB within a single insect. A detection of ca. 50 novel species within microbiota to their honey and corbicular pollen. In this study, 8 these bacterial genera will make a huge impact in their current we found the highest concentration of viable LAB (10 per gram . The findings of L. kunkeei as common symbionts with honey) in Nepalese honey of A. laboriosa and similar quantities in A. Apis and stingless bees highlight the importance of this organism. mellifera honey from Africa (Table 1). We have previously shown the consistent presence and dominance of this lactobacilli, in our studies of A. mellifera (25% of 158 isolates Ontogeny and maintenance of LAB in Sweden [16,17,19]; 40% of 42 isolates in USA [18]; 28% of 50 The results were clear-cut; at eclosion, crops were empty and isolates in Africa [17], and now in all Apis spp. and in the stingless devoid of LAB; but within minutes post-eclosion the microbiota bees. The most recent common ancestor to honeybees and builds up gradually by trophallactic exchange with nestmates stingless bees has been dated to .80 million years ago [27], (Table 2). L. kunkeei was found to dominate the crop microbiota at suggesting that the L. kunkeei-dominated LAB flora is an ancient all sampling occasions. Only six honeybee crop LAB members apine bee association. But invariance in L. kunkeei 16S rRNA gene were found during the trial. Honeybees kept in solitary sequences across host species and geographic locality (Figure 1) confinement from eclosion (n = 10) retained sterile crops. suggest possible horizontal transfer of LAB members between host The LAB microbiota remains viable within bees and added in species. However, in Borneo the five sympatric Apis spp. forage high concentration to their food products (Table 1) as the together with Trigona spp. on the same flowers but L. kunkeei was symbionts detach from their niche the honey crop. We observed not found in any Trigona spp. investigated, arguing against by in vitro and in vivo investigations of the LAB symbionts with SEM horizontal transfer.

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Figure 1. Phylogenetic tree of LAB in bee crops. Phylogenetic tree based on a distance matrix of positions 56–1470 (Escherichia coli numbering) in the 16S rRNA gene of Lactobacillus and Bifidobacterium spp. bacteria from Apis species (red) and the stingless bees Melipona beechii (turquoise), Meliponula bocandeii (green) and Trigona sp (blue). Previously characterized bacterial phylotypes from Apis mellifera are in bold print (red). Bacterial type strains are in bold characters (black). Phylotypes in the framework (right) represent different clusters belonging to a characterised or possibly novel species. Bifidobacterium group is the out-group. Bar: 5 base pair changes. 16S rRNA gene sequences deposited in GenBank HM534742– HM534842 (in parenthesis). doi:10.1371/journal.pone.0033188.g001

Our investigation shows how this microbiota is acquired produced by food grade organisms (GRAS), in particular LAB, (Table 2) and maintained within bees (Figure 2 and Figure 3; may confer health benefits in humans [32,33,34]; the same may be Video S1 and Video S2). Interestingly, LAB builds up gradually by true for honeybees. The complexity of attachment and biofilm trophallactic exchange with nestmates and L. kunkeei Fhon2 was formation by this symbiotic community comprise yet unknown found to dominate the crop microbiota at all sampling occasions mechanisms of action. These may include membrane properties of again reflecting its importance (Table 2). However, that only six the symbionts to avoid rejection by their host, as well as the honeybee crop LAB members were found during the trial may production of potent antimicrobial substances. reflect either the disadvantage for bacterial counting of using During foraging, foreign microbes are introduced into bees and viable counts that display the dominant bacteria or the numerical to their colony through their honey crop, with collected nectar, variation across seasons. We know from our previous studies that and through pollen (Table S1). When a flower blooms for the first the LAB members in the crop vary numerically across seasons with time, its nectar and pollen are sterile but eventually become the flowers visited by bees and with the health status of bees [16]. invaded by airborne microorganisms and microbes from insects. On the other hand, we know that the microbiota is also rather The composition and numbers of this transient microbiota may consistent across Apis species. At first sight, it is surprising how this vary with time but also with flower type, visiting insects, microbiota is maintained within the honey crop, with the extensive temperature and the nutritional composition of the pollen and ebbing and flowing of sugars, enzymes, water and the constant nectar. LAB members in the crop vary numerically [16] but are invasion of foreign microbes following ingestion of flower nectar consistent within the same Apis species [17]. LAB diversity could during foraging. Visualization of the crop and attached LAB be explained by variation in nutrient content of different nectars reveals how this microbiota is conserved (Figure 2 and Figure 3; and pollen and also by variation in the microbes to which flowers Video S1 and Video S2) in networks and biofilms. The property of are exposed. Transient floral microbes may trigger the growth of biofilm formation is known in LAB species that resides in the resident LAB microbiota in bees and their production of human gut and vagina [28,29,30]. In addition to the well- antimicrobial substances, a mechanism known for LAB strains in described characteristic of LAB to produce exopolysaccharides, other niches (e.g. Lactobacillus reuteri when producing reuterin other likely mechanisms in biofilm formation and adhesion include [35,36,37]. the production of proteins, carbohydrates, enzymes, nucleic acids, We raised the hypothesis that honeybee LAB possess antimi- lipids or membrane bound receptors. Exopolysaccharides are the crobial properties against microorganisms present in nectars and main component in extracellular polymeric substances (EPS) and on pollen in order to defend their niche (the honey crop) and when, secreted into the environment, provide protection to prevent spoilage of honey and bee bread during their production, bacteria; they are also involved in host colonization and cellular which may take from days to weeks. Our results show a recognition [31]. It has been suggested that exopolysaccharides preliminary overall inhibition of transient environmental microbes

Table 1. Lactic acid bacterial counts in social bees.

Bee species honey crop honey (g21) bee pollen (g21) bee bread (g21)

Apis melliferaab 106 107 108 106 Apis nuluensisb 106 NDc 106 NDc Apis nigrocinctab 107 105 109 105 Apis koschevnikovi 105 106 NDc 105 Apis cerana 106 105 106 105 Apis andreniformisb 103 104 106 104 Apis floreab 106 102 106 NDc Apis laboriosab 106 108 NDc 109 Apis dorsatab 105 NDc 106 NDc Melipona beecheiib 107 105 106 106 Meliponula bocandeib 107 103 106 107 Trigona sp.b 105 104 107 104

aApis mellifera bees collected in Kenya from the subspecies A. m. scutellata and A. m. monticola. bWild colonies. cND = Not determined. Legend. Viable counts in colony forming units (cfu) of lactic acid bacteria cultivated from honey crops, fresh honey, bee pollen and bee bread. All samples were cultivated on LAB selective media immediately after sampling or after chilled transportation. doi:10.1371/journal.pone.0033188.t001

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Table 2. Presence of LAB in callows.

LAB At eclosiona 1 h 24 h 3 days 1 week 2 weeks

Bma5 - - - 1 2 3 Hma8 - 1 - - - 1 Biut2 ------Hma2 ------Hma11 ------Hon2 - - - - - 1 Bin4 ------Fhon13 ------Fhon2 - 17 6 16 13 9 Bma6 ------Bin7 ------Figure 3. In vivo visualization of LAB biofilm. The red fluorescence shows live-stained bacteria in a LAB biofilm attached to a honey crop. Hma3 - - 1 - - 1 The green fluorescence shows the nuclei of the honeybee crop cells. Bin2 - - - 1 1 - The visualized tissue shows a projection of 112 confocal z-sections (through a 37.6 mm z-depth, covering a xy-area of 2466246 mm). aDisplayed numbers represent randomly picked isolates. doi:10.1371/journal.pone.0033188.g003 Legend. Ten to 30 colonies were randomly picked from agar plates containing 30–300 agents including common organic acids, proteins, peptides, colonies each, and re-cultivated for purity (isolates). The re-cultivation and enzymes, and bacteriocins that we are currently characterising. identification of LAB were performed as previously described [16]. doi:10.1371/journal.pone.0033188.t002 The LAB microbiota of the A. mellifera honey crop is added by bees to their brood food and corbicular pollen and is important in the production of honey and bee-bread [16,19]. It is well known that found in flowers (Table S1). Once again, L. kunkeei, the most species within Lactobacillus and Bifidobacterium inhibit pathogens; they common species in all Apis bees (Figure 1), was highlighted as the have been used for centuries in food preservation to prevent most potent, inhibiting all tested microorganisms. This bacterium microbial spoilage [40]. Commercial probiotic products for human was originally described as a wine-spoiling organism since it consumption with viable LAB contain about the same quantity (108) inhibited yeast in wine production [38,39]. Interestingly, the of mostly one single LAB g21 product [41,42]. We hypothesise that extent of inhibition by single LAB members varied considerably the resident strains of Lactobacillus and Bifidobacterium in honey could with test microbes. Yet the LAB microbiota seems to work in a function in a similar way as LAB for food preservation or as a synergistic matter [24] (Table S1); they produce antimicrobial defence against microorganisms invading humans.

Figure 2. In vitro visualization of lactobacilli. In vitro visualization of lactobacilli attached to the wall of a honeybee crop using SEM. A, B and C show different areas of a honey crop at similar magnification with visible attachment structures resembling extracellular polymeric substances (EPS). D shows a larger part of the crop with attached bacteria. Photographer Lennart Nilsson. doi:10.1371/journal.pone.0033188.g002

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Figure 4. LAB rescue of honeybee larvae from European Foulbrood. Proportion dead larvae in both replicates (total number dead after 21 days). Data presented as a mean for the three groups fed M. plutonius (107,106 and105 bacteria ml21), and the 3 groups fed M. plutonius and LAB. Irrespective of infectious dose, the overall effect from the LAB supplement was a significantly reduced mortality in the treated groups. Mortality in the uninfected control groups was ,7%. doi:10.1371/journal.pone.0033188.g004

Honey collected from managed or wild colonies of Apis spp. or In order to secure honeybee pollination services, A. mellifera stingless bees has been independently regarded as a therapeutic beekeepers replace harvested honey by feeding sugar solutions, agent by many cultures throughout history, from the Maya in occasionally mixed with antibiotics for prophylactic control of Mexico to the Pharaohs in Egypt [43], possibly reflecting honeybee-specific bacterial diseases of bee brood such as AFB and beneficial effects of the viable honeybee microbiota when microsporidia [47]. It is known that LAB antibiotic susceptibility consumed or applied on wounds. It is feasible to believe that varies [48,49]. In vitro culturing of the 13 Apis individual LAB fresh honey represents a naturally occurring probiotic product, members with two antibiotics used in apiculture (oxytetracycline one with a great diversity and concentration of LAB species and tylosin) demonstrated high sensitivity of all to Tylosin, the (Table 1) that may reflect a myriad of beneficial properties of every most recently employed antibiotic within apicultural practices in specific LAB member in the honey crop. the USA [26]. Nevertheless, strains L. kunkeei Fhon2 and We believe that LAB antimicrobial mechanisms have evolved in Lactobacillus Fhon13, Hma11, Hma8 and Hon2 showed resistance synergy with bees to defend themselves and their hosts from to oxytetracycline that may reflect the extended use of this environmental threats such as microbes found in nectars and antibiotic in apiculture or their long-term exposure to environ- pollen, and possibly for defence against specific honeybee mental microbes from the surrounding environment that produce pathogens. This ancient symbiotic relationship between LAB similar substances. The negative effects on honeybee health from and bees seems to be of great benefit for bees and may be referred damaging the honey crop microbiota by the use of these antibiotics to as colonization resistance. The same phenomenon described for need to be investigated further. the normal flora in the fruit fly gut [6]. Honeybee brood are fed bee-bread containing viable LAB and their antimicrobial sub- Perspectives stances. Thus, our results strongly suggest that LAB linked to the The economic value of commercial honeybee pollination is honeybee crop have important implications for honeybee estimated at over US $14 billion in the USA alone and over US pathology, particularly for bacterial brood diseases such as AFB $220 billion worldwide [50]. Yet ongoing colony losses in the USA and EFB. Honeybees are considered to have only about a third of and Europe defy causal explanation despite intensive research the innate immune genes compared to other insects [44,45]. In effort [51,52] and identification of emergent and exotic pathogens addition to social defences that accrue to social insects [46], [20]. Our discovery of a diverse and novel honey crop LAB individual honeybees may also benefit from their LAB symbionts, microbiota common to all recognized honeybee species plus 3 which are probably of great importance in pathogen defence, stingless bee species may be the missing link in this worldwide possibly further reducing dependency on the innate immune problem. Since related microbiotas are found across bee species, it system. strongly suggests a close evolutionary relationship between

PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e33188 Symbiosis between LAB and Honeybees bacteria and hosts, as well as underscoring the importance of LAB Ontogeny of LAB symbionts for bees. Not only are LAB symbionts involved in To determine how the crop microbiota is acquired, we marked honeybee food production and preservation, they are also of Western honeybees (A. mellifera) at eclosion from their wax brood importance in host defence against pathogen and transient cells (n = 30), returned them to the hive, collected them at different microbes intercepted during foraging. The importance of this ages, cultivated the contents of their crops and identified the LAB crop microbiota for honeybees is additionally strengthened by the (cultivation and identification as described previously [16]). fact that it is immediately transferred to the sterile crop of newly emerged bees by trophallactic exchange with nestmates. Any beneficial effect from this microbiota may be undermined Maintenance of the honey crop microbiota where prophylactic use of antibiotics is practiced (e.g. USA) or We performed in vitro and in vivo investigations of the symbionts where their natural foodstuffs, honey and pollen, are supplement- with SEM and fluorescence microscopy. The SEM samples were ed by the beekeeper with synthetic sugars and pollen substitutes prepared by freeze drying [53] and pictures were taken by lacking LAB or their beneficial substances. The absence of LAB is Photographer Lennart Nilsson (Sweden). especially problematic when the bees attempt to produce and The preparation and confocal fluorescence microscopy of preserve food for themselves and their brood, when feeding their bacteria in the honey crop was achieved as follows. Honeybees brood with pollen lacking LAB or LAB derived antimicrobial were fed with a mixture of honey and water containing (SytoxH, substances, when nestmates establish a LAB microbiota in callows Green dye and BacLightTM, Red bacterial stain, Molecular by trophallactic exchange, or when pathogens invade their hive. Probes) to discriminate the cells of the bee from the living bacteria. Emphasis now needs to be given to discovering the mechanisms of Following an incubation of approx. 15 min, the honey crop was action of LAB against pathogens and food spoiling microbes, and dissected at room temperature. The crop was opened with a single how they can be used to resolve ongoing honeybee colony losses, longitudinal cut. To prevent the contraction of the muscles of the in which LAB may be the important missing link. Altered crop wall, the crop was rinsed in phosphate buffered saline (PBS) beekeeping husbandry practices that enhance LAB are needed, or supplemented with 1 mM EDTA and mounted on a glass slide. direct manipulation by supplementary feeding of individual or Slides were examined using a TCS SP5 laser confocal microscope composite LAB members and their products could help alleviate equipped with continuous spectrum white laser (Leica, Mannheim CCD. Further functional analysis of LAB in bees will certainly Germany). The images were captured using a 636 oil immersion enrich our understanding of insect-microbe symbioses and their objective (NA 1.4 HCX PL APO CS) with filter setup adapted to evolutionary dynamics within complex eusocial insect societies. FITC and Texas Red dual colour illumination. The raw images were processed in ImageJ (NIH, Bethesda, USA) using median Materials and Methods filtering. The Z-stacks was visualized using the ImageJ plugin 3D- viewer. The 3D-Movies (Video S1 and Video S2) show a Ethics projection of 90 confocal z-sections through a z-depth of No specific permits were required for the described field studies. 45.3 mm, covering an area of 2466246 mm in the xy-direction. Local colleagues (described in the acknowledgments) collected samples where permission was not required i.e. not from nature reserves or privately-owned locations. The field studies did not Melissococcus plutonius bioassay involve endangered or protected species. We investigated possible inhibitory effects on European foulbrood (EFB), a major bacterial pathogen of larval honeybees, LAB diversity in bees from the LAB microbiota using both in vitro and in vivo tests, as We sampled the honey crop LAB microbiota of all 9 well previously described [24]. As adult honeybee workers feed larvae recognized honeybee (Apini) species plus 3 stingless bee species with crop contents, this represents a typical means by which (Meliponini). Apis andreniformis (n = 3 colonies), Apis cerana (n = 2 larval food acquires LAB. Bacterial suspensions of Melissococcus colonies), Apis koschevnikovi (n = 3 colonies), Apis nuluensis (n = 1 plutonius (provided by Dr. Jean-Daniel Charrie`re, Agaroscope, colony) and Apis dorsata (n = 1 colony) were collected from Borneo Switzerland, Accession nr: JN689233) were prepared fresh for each experiment and diluted in larval food for final concentra- (Malaysia), Apis laboriosa (n = 2 colonies) from Nepal, Apis florea 7 6 5 (n = 1 colony) from Thailand, Apis nigrocincta (n = 1 colony) from tions of 10 ,10 and 10 bacteria per ml. A mixture of the Indonesia, and A. mellifera (n = 25 colonies) from Sweden and thirteen previously described honeybee LAB [16,17,18] in Kenya. Samples of the stingless bee genera Trigona were collected approximately equal proportions was diluted in larval food for 7 from Thailand (n = 1 colony) and Borneo (n = 1 colony) (Malaysia), a final, total concentration of 10 LAB per ml. A. mellifera worker Meliponula bocandeei (n = 2 colony) from Kenya, and larvae were grafted and reared in vitro [24]. Briefly, first instar (n = 2 colonies) from Mexico. For each bee species, the honey crop worker larvae were transferred to the surface of the larval diet of LAB content of 10–20 bees was analysed. For A. nuluensis and A. the different treatments. i) control group provided with dorsata we only analysed the honey crop content and the corbicular uninfected diet, ii) control group initially fed uninfected diet but pollen from field collected bees as we were unable to sample LAB supplemented food after 48 hours onwards and iii) colonies of these free-living bees. Isolation of LAB from honey experimental groups provided larval diet spiked with defined 7 6 5 21 crops, fresh honey, corbicular bee pollen and bee bread was amounts of M. plutonius (10 ,10 and 10 bacteria ml ). Twenty- performed as previously described [16,19]. PCR-amplification of four hours post exposure; larvae were transferred to wells isolates for 16S rRNA gene sequencing, identification and containing uninfected diet and LAB supplemented food 48 hours phylogenetic analysis were performed according to Olofsson and post-infection onwards. The experiment was finished 21 days Va´squez [16]. Additionally, the 16S rRNA gene sequences were post-infection and larval mortality was monitored daily. A total of also checked against the software RDP (Ribosomal Database 420 larvae were used in two replicate experiments. The PoloPlus Project II), accessible from the homepage (http://rdp.cme.msu. Probit and Logit Analysis program (version 2.0, LeOra software) edu/). A total of 750 lactic acid bacterial isolates were retrieved in was used to compare mortality rates between the experimental this study. groups in the exposure bioassay.

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Flowers, nectar, pollen and microorganisms mellifera bees in Sweden. Fifty five different species of We analysed the microbial composition of 15 flowers frequently bacteria and 5 yeast species from the flowers were analysed in vitro visited by A. mellifera in Sweden (Table S1). Flowers were collected against all 13 honey crop LAB from A. mellifera grown individually aseptically in Kullaberg, Sweden. The flowers were then shaken in and together (right column). Inhibition zones are displayed as sterile buffer (PBS) and immediately transported to the Laboratory diameter in millimetres. Zero indicates no inhibition and (-) at Lund University. Dilutions were made with sterile peptone indicates no result. water (0.2% w/v), spread on MRS (Oxoid), APT (Oxoid) and TSB (DOC) (Oxoid) agar plates incubated anaerobically at 35uC (MRS and Video S1 A biofilm formed by the LAB microbiota is APT agar plates) and aerobically at 22uC (TSB agar plates) during seen as a red carpet and the confocal z-stack reveals that 5 days. Identification of the microbial isolates was achieved by the bacteria are located in crypts all through the z-depth sequencing the 16S rRNA genes (for bacteria) [16] and the D1-D2 of the crop wall. regions of the LSU 26S rRNA genes (for yeasts) [54]. (AVI) Dual-culture overlay assay Video S2 A 3D-view of a typical biofilm is shown in a We analysed the inhibition properties of all honey crop LAB 360 degree rotation of a projection of 90 confocal z- grown individually and together against the pathogen M. plutonius sections through a z-depth of 45.3 mm. and also against the 55 bacterial strains and 5 yeast strains isolated (AVI) from flowers. The assays were performed as earlier described [55] with the following modifications. LAB were inoculated on MRS Acknowledgments agar plates (Oxoid, supplemented with 0.1% L-cysteine and 2.0% fructose) during 12 hours. We used the medium previously The authors thank photographer Lennart Nilsson and the city authorities described for cultivation of M. plutonius [56,57] and the same of Helsingborg that supported his work. We are grateful for the bee samples provided by Luis Medina, Mexico; Farooq Ahmad, Nepal; media as for the isolation of microbes from flowers for the over Mananya Phaincharoen, Thailand, Mappatoba Sila, Indonesia; and layer of soft agar. After an incubation of 2–4 days depending on Joseph Macharia, Kenya. We are thankful for the time we spent on growth rates the zone of inhibition was measured. Borneo at the Agricultural Research Station, Tenom, Malaysia, working with Hubert Lim. LAB and antibiotics Honey crop LAB were tested for susceptibility to oxytetracycline Author Contributions and tylosin by disc diffusion on MRS agar (Oxoid, supplemented Conceived and designed the experiments: TCO AV. Performed the with 0.1% L-Cysteine and 2.0% fructose). experiments: TCO AV IF E. Forsgren E. Flaberg LS. Analyzed the data: AV TCO E. Forsgren IF RJP E. Flaberg LS. Contributed reagents/ Supporting Information materials/analysis tools: RJP. Wrote the paper: AV RJP TCO. Table S1 The microbial composition of the flowers of 15 Angiosperma (left column) frequently visited by Apis

References 1. Rajilic-Stojanovic M, Smidt H, de Vos WM (2007) Diversity of the human 14. Ventura M, O’Flaherty S, Claesson MJ, Turroni F, Klaenhammer TR, et al. gastrointestinal tract microbiota revisited. Environ Microbiol 9: 2125–2136. (2009) Genome-scale analyses of health-promoting bacteria: probiogenomics. 2. Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, et al. (2005) Nat Rev Micro 7: 61–71. Diversity of the human intestinal microbial flora. Science 308: 1635–1638. 15. Euze´by JP List of Prokaryotic names with Standing in Nomenclature - Genus 3. Backhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI (2005) Host- Lactobacillus. Available: http://www.bacterio.cict.fr/l/lactobacillus.html. Ac- bacterial mutualism in the human intestine. Science 307: 1915–1920. cessed 2012 Jan 5. 4. Dillon RJ, Dillon VM (2004) THE GUT BACTERIA OF INSECTS: 16. Olofsson TC, Va´squez A (2008) Detection and Identification of a Novel Lactic Nonpathogenic Interactions. Annual review of entomology 49: 71–92. Acid Bacterial Flora Within the Honey Stomach of the Honeybee Apis mellifera. 5. Ryu J-H, Kim S-H, Lee H-Y, Bai JY, Nam Y-D, et al. (2008) Innate Immune Current microbiology 57: 356–363. Homeostasis by the Homeobox Gene Caudal and Commensal-Gut Mutualism 17. Olofsson TC, Va´squez A, Sammataro D, Macharia J (2011) A scientific note on in Drosophila. Science 319: 777–782. the lactic acid bacterial flora within the honeybee subspecies; Apis mellifera 6. Silverman N, Paquette N (2008) Immunology. The right resident bugs. Science (Buckfast), A. m. scutellata, A. m. mellifera,andA. m. monticola. Apidologie. pp 1–4. 319: 734–735. 18. Va´squez A, Olofsson TC, Sammataro D (2009) A scientific note on the lactic 7. Evans J, Armstrong T-N (2006) Antagonistic interactions between honey bee acid bacterial flora in honeybees in the USA – A comparison with bees from bacterial symbionts and implications for disease. BMC Ecology 6: 4. Sweden. Apidologie 40: 26–28. 8. Hughes DP, Pierce NE, Boomsma JJ (2008) Social insect symbionts: evolution in 19. Va´squez A, Olofsson TC (2009) The lactic acid bacteria involved in the homeostatic fortresses. Trends Ecol Evol 23: 672–677. production of bee pollen and bee bread. Journal of Apicultural Research 48: 9. Hammes W, Hertel C (2006) The Genera LactobacillusandCarnobacterium: The 189–195. Prokaryotes. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, 20. Cox-Foster D, Sean C, Edward CH, Gustavo P, Jay DE, et al. (2007) A Stackebrandt E, eds. Springer New York. pp 320–403. Metagenomic Survey of Microbes in Honey Bee Colony Collapse Disorder. 10. FAO/WHO (2001) Health and nutritional properties of probiotics in food Science 318: 283–287. including powder milk with live lactic acid bacteria, a joint FAO/WHO expert 21. van Engelsdorp D, Evans JD, Saegerman C, Mullin C, Haubruge E, et al. (2009) consultation Cordoba, Argentina. Available: http://www.who.int/foodsafety/ Colony Collapse Disorder: A Descriptive Study. PLoS ONE 4: 1–17. publications/fs_management/probiotics/en/index.html#.TmTPiQL25zM. 22. Martinson Vincent G, Danforth Bryan N, Minckley Robert L, Rueppell O, email. Accessed 2012 Jan 5. Tingek S, et al. (2011) A simple and distinctive microbiota associated with honey 11. FAO/WHO (2002) Guidelines for the Evaluation of Probiotics in Food:Joint bees and bumble bees. Molecular Ecology 20. FAO/WHO Working Group meeting. London Ontario, Canada. Available: 23. Mohr Kathrin I, Tebbe Christoph C (2006) Diversity and phylotype consistency http://www.who.int/foodsafety/publications/fs_management/probiotics2/en/ of bacteria in the guts of three bee species Apoidea at an oilseed rape field. index.html. Accessed 2012 Jan 5. Environmental Microbiology 8: 258–272. 12. Klaenhammer T, Altermann E, Arigoni F, Bolotin A, Breidt F, et al. (2002) 24. Forsgren E, Olofsson TC, Va´squez A, Fries I (2010) Novel lactic acid bacteria Discovering lactic acid bacteria by genomics. Antonie van Leeuwenhoek 82: inhibiting Paenibacillus larvae in honey bee larvae. Apidologie 41: 99–108. 29–58. 25. Ludwig W, Strunk O, Klugbauer S, Klugbauer N, Weizenegger M, et al. (1998) 13. Servin AL (2004) Antagonistic activities of lactobacilli and bifidobacteria against Bacterial phylogeny based on comparative sequence analysis. Electrophoresis microbial pathogens. FEMS Microbiology Reviews 28: 405–440. 19: 554–568.

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26. FDA (2005) FDA Approves TYLAN Soluble for the Control of American cultures and in food matrices. International Journal of Food Microbiology 149: Foulbrood in Honey Bees. Available: http://www.fda.gov/AnimalVeterinary/ 185–193. NewsEvents/CVMUpdates/ucm048398.htm. Accessed 2012 Jan 5. 42. Gorbach S, Seppo S, Yuan-Kun L, Benno Y (2004) Human Studies on 27. Cardinal S, Straka J, Danforth BN (2010) Comprehensive phylogeny of apid Probiotics. Lactic Acid Bacteria: CRC Press. bees reveals the evolutionary origins and antiquity of cleptoparasitism. 43. Crane E The world history of beekeeping and honey hunting: Routledge. Proceedings of the National Academy of Sciences 107: 16207–16211. 44. Consortium THGS (2006) Insights into social insects from the genome of the 28. Domingue PA, Sadhu K, Costerton JW, Bartlett K, Chow AW (1991) The honeybee Apis mellifera. Nature (London) 443: 931–949. human vagina: normal flora considered as an in situ tissue-associated, adherent 45. Evans JD, Lopez DL (2004) Bacterial Probiotics Induce an Immune Response in biofilm. Genitourinary Medicine 67: 226–231. the Honey Bee (Hymenoptera: Apidae). Journal of Economic Entomology 97: 29. Lebeer S, Verhoeven TLA, Ve´lez MP, Vanderleyden J, De Keersmaecker SCJ 752–756. (2007) Impact of Environmental and Genetic Factors on Biofilm Formation by 46. Wilson-Rich N, Spivak M, Fefferman NH, Starks PT (2009) Genetic, Individual, the Probiotic Strain Lactobacillus rhamnosus GG. Applied & Environmental and Group Facilitation of Disease Resistance in Insect Societies. Annual review Microbiology 73: 6768–6775. of entomology 54: 405–423. 30. Macfarlane S, Dillon JF (2007) Microbial biofilms in the human gastrointestinal 47. Morse RA, Hooper T The Illustrated encyclopedia of beekeeping: E.P. Dutton. tract. Journal of Applied Microbiology 102: 1187–1196. 48. Ammor MS, Belen Florez A, Mayo B (2007) Antibiotic resistance in non- 31. Flemming H-C, Wingender J (2010) The biofilm matrix. Nat Rev Micro 8: enterococcal lactic acid bacteria and bifidobacteria. Food Microbiology 24: 623–633. 559–570. 32. De Vuyst L, De Vin F, Vaningelgem F, Degeest B (2001) Recent developments 49. Moubareck C, Gavini F, Vaugien L, Butel MJ, Doucet-Populaire F (2005) in the biosynthesis and applications of heteropolysaccharides from lactic acid Antimicrobial susceptibility of bifidobacteria. Journal of Antimicrobial Chemo- bacteria. International Dairy Journal 11: 687–707. therapy 55: 38–44. 33. Jolly L, Vincent SJF, Duboc P, Neeser J-R (2002) Exploiting exopolysaccharides 50. Gallai N, Salles J-M, Settele J, Vaissie`re BE (2009) Economic valuation of the from lactic acid bacteria. Antonie van Leeuwenhoek 82: 367–374. vulnerability of world agriculture confronted with pollinator decline. Ecological 34. Jones S, Versalovic J (2009) Probiotic Lactobacillus reuteri biofilms produce Economics 68: 810–821. antimicrobial and anti-inflammatory factors. BMC Microbiology 9: 35. 51. Moritz RFA, de Miranda J, Fries I, Le Conte Y, Neumann P, et al. (2010) 35. Axelsson LT, Chung TC, Dobrogosz WJ, Lindgren SE (1989) Production of a Research strategies to improve honeybee health in Europe*. Apidologie 41: Broad Spectrum Antimicrobial Substance by Lactobacillus reuteri. Microbial 227–242. Ecology in Health and Disease 2: 131–136. 52. Pettis JS, Delaplane KS (2010) Coordinated responses to honey bee decline in 36. Chung TC, Axelsson L, Lindgren SE, Dobrogosz WJ (1989) In Vitro Studies on the USA*. Apidologie 41: 256–263. Reuterin Synthesis by Lactobacillus reuteri. Microbial Ecology in Health and 53. Bell PB, Jr., Lindroth M, Fredriksson BA (1988) Preparation of cytoskeletons of Disease 2: 137–144. cells in culture for high resolution scanning and scanning transmission electron 37. Talarico TL, Dobrogosz WJ (1989) Chemical characterization of an microscopy. Scanning Microscopy 2: 1647–1661. antimicrobial substance produced by Lactobacillus reuteri. Antimicrob Agents 54. Cano J, Guarro J, Gene´ J (2004) Molecular and Morphological Identification of Chemother 33: 674–679. Colletotrichum Species of Clinical Interest. Journal of clinical microbiology 42: 38. Edwards CG, Haag KM, Collins MD, Hutson RA, Huang YC (1998) 2450–2454. Lactobacillus kunkeei sp. nov.: a spoilage organism associated with grape juice 55. Magnusson J, Stro¨m K, Roos S, Sjo¨gren J, Schnu¨rer J (2003) Broad and fermentations. Journal of Applied Microbiology 84: 698–702. complex antifungal activity among environmental isolates of lactic acid bacteria. 39. Huang Y-C, Edwards CG, Peterson JC, Haag KM (1996) Relationship Between FEMS Microbiology Letters 219: 129–135. Sluggish Fermentations and the Antagonism of Yeast by Lactic Acid Bacteria. 56. Bailey L, Collins MD (1982) Reclassification of ‘Streptococcus pluton’ (White) in a Am J Enol Vitic 47: 1–10. new genus Melissococcus,asMelissococcus pluton nom. rev.; comb. nov. Journal of 40. Maarit M (2004) Lactic Acid Bacteria in Vegetable Fermentations. Lactic Acid Applied Microbiology 53: 215–217. Bacteria: CRC Press. 57. Hornitzky MAZ, Smith L (1998) Procedures for the culture of Melissococcus pluton 41. Champagne CP, Ross RP, Saarela M, Hansen KF, Charalampopoulos D (2011) from diseased brood and bulk honey samples. Journal of Apicultural Research Recommendations for the viability assessment of probiotics as concentrated 37: 293–294.

PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e33188